Conventional wisdom predicted that the majority of previously healthy patients who developed acute kidney injury (AKI) would recover without significant renal sequelae. However, recent large studies indicate that even healthy patients are at significant risk of developing chronic kidney disease (CKD) and end-stage renal disease (ESRD) after one episode of AKI (Chawla et al., 2011; Lo et al., 2009; Amdur et al., 2009; Wald et al., 2009; Chawla and Kimmel, 2012). An Ontario study examined over 41,000 patients who survived an episode of AKI without requiring acute dialysis at the time of the AKI (Wald et al., 2012). Compared to matched control patients, the patients with AKI had a near 3-fold increase in late ESRD necessitating chronic dialysis (Wald et al., 2012). Thus, even a single episode of what would previously have been regarded as “mild” AKI sets the stage for later CKD and ESRD.
Ischemia-reperfusion-injury (IRI) is a cause of AKI and may progress to CKD (also known as chronic renal injury) as a result of progressive renal fibrosis, in which normal elements of the renal tubulointerstitium are replaced by myofibroblasts that secrete collagenous extracellular matrix (Bonventre and Yang, 2011; Quaggin and Kapus, 2011; Venkatachalam et al., 2010). The main features include recruitment and proliferation of myofibroblasts which secrete collagenous extracellular matrix, and loss of capillary density (Zeisberg and Neilson, 2010). Eventually, the normal tubular and vascular structures of the renal interstitium undergo atrophy and become replaced by fibrous scar. Once recruited to the tubulointerstitium, pericytes become myofibroblasts. In response to locally produced fibrogenic cytokines and growth factors, especially transforming growth factor beta (TGF-β), myofibroblasts proliferate and secrete abundant quantities of extracellular matrix proteins, particularly collagen type I and fibronectin (Kida and Duffield, 2011; Schrimpf and Duffield, 2011; Lin et al., 2011). The net result is replacement of functional nephrons by scar tissue and progressive kidney failure.
Diabetes is the leading cause of chronic kidney disease in most countries around the world [Rossing 2006]. In the United States alone, it is estimated that nearly $17 billion is spent annually on diabetic nephropathy care [Gordois et al. 2004]. While glycemic control [Diabetes Control and Complications Trial Research Group 1993, UKPDS 1998], blood pressure regulation [Adler et al. 2000], and renin-angiotensin system blockade [Lewis et al. 2001; Brenner et al. 2001; Lewis et al. 1993] slow nephropathy progression, many patients still progress to kidney failure, a costly and life-threatening condition requiring renal replacement therapy. New treatments are clearly needed.
Diabetic nephropathy is driven by a complex set of inter-related pathways. Early on, diabetes is commonly characterized by hyperfiltration, a phenomenon that has been linked epidemiologically [Costacou et al. 2009, Magee et al. 2009; Ruggenenti et al. 2012] and mechanistically [Anderson et al. 1986] to poor long-term renal outcomes. While classically felt to be driven by altered renal hemodynamics [Anderson et al. 1993, Sochet et al. 2006], emerging evidence suggests that glomerular angiogenesis also augments GFR through increases in filtration surface area created by nascent capillaries [Osterby et al. 1988, Hirose et al. 1980]. Later, diabetic nephropathy is characterized by fibrosis, a largely irreversible process that obliterates both glomeruli and the tubulointerstitium [Gilbert et al. 1999]. Transforming growth factor-β (TGF-β) is a central driver of diabetic fibrogenesis, activating a variety of pro-fibrotic signaling pathways. In particular, two TGF-β signaling intermediates, the Receptor Smads (Smad2 and Smad3) and RhoA, mediate fibrogenesis via both independent and inter-related mechanisms [Engel et al. 1999; Bhowmick et al. 2001]. While anti-TGF-β therapies may block fibrosis, they also inhibit other critical non-fibrogenic TGF-β effects, including its potent immunosuppressive activity. Thus, anti-TGF-β blockade has been a largely unsuccessful treatment strategy for diabetic nephropathy.
The Slit family of proteins, together with their transmembrane receptor, Roundabout (Robo), were initially shown to repel axons and neuronal migration during development of the central nervous system (CNS) (Brose et al., 1999; Kidd et al., 1999). There are three known mammalian Slit family members. Slit1 is predominantly expressed in the CNS, while Slit2 and, to a lesser degree, Slit3 are expressed in other tissues, especially kidney, heart, and lung (Wu et al., 2001). Slit protein expression persists into adulthood, suggesting roles beyond those during development. Slit proteins are structurally unique, having both epidermal-like growth factor and leucine-rich repeats (LRR). These features allow secreted Slit to interact with varied proteins, including cell surface receptors and extracellular matrix proteins such as glypican-1 (Ronca et al., 2001). Thus, Slit can act as a local, non-diffusible signaling molecule. Proteolytic cleavage of Slit2, perhaps by metalloproteases, generates N- and C-terminal fragments (Slit-N and Slit-C) (Brose et al., 1999; Schimmelpfeng et al., 2001; Wang et al., 1999). Slit2-N is sufficient to engage its receptor and to induce signaling (Nguyen et al., 2001; Battye et al., 2001; Chen et al., 2001).
Robo is a single-pass type 1 transmembrane protein. The extracellular region contains five immunoglobulin (Ig)-like domains and three fibronectin type III repeats while the intracellular domain contains four conserved cytoplasmic motifs (CC0, CC1, CC2, and CC3). The extracellular Ig-like domains of Robo are sufficient for binding the LRR of Slit. The intracellular CC3 motif is necessary for the repulsive response to Slit. Mammals have four Robo isoforms, of which Robo-1 is most widely expressed in non-neural cells, especially immune cells (Wu et al., 2001; Prasad et al., 2007; Tole et al., 2009).
After Slit2 binds to the extracellular domain of Robo-1, the intracellular domain of Robo-1 associates with a novel family of GTPase activating proteins (GAPs), namely Slit-Robo GAPs (srGAP) (Wong et al., 2001). By preventing activation of Cdc42, Slit2 inhibited migration of cells from the anterior subventricular zone of the forebrain ((Wong et al., 2001). Recent studies examined the effects of Slit2 on migration of vascular smooth muscle cells (VSMC), lymphocytes, and neutrophils towards platelet-derived growth factor B (PDGF), the chemokine CXCL12, and formyl-methionyl-leucyl-phenylalanine (fMLP), respectively (Prasad et al., 2007; Tole et al., 2009; Liu et al., 2006). These chemoattractants induce cell migration by activating Rac, Cdc42, and/or Rho, crucial for reorganization of the cytoskeleton. Slit2 inhibited activation of Rac, Cdc42 and/or Rho, and consequent migration of VSMC, lymphocytes, and neutrophils (Prasad et al., 2007; Tole et al., 2009; Liu et al., 2006). The present inventors and others also showed that Slit2 inhibits migration of leukocytes and cancer cells, and inhibits platelet adhesion by preventing activation of Akt and Erk MAPK (Patel et al., 2012; Prasad et al., 2007; Tole et al., 2009; Prasad et al., 2004).
During kidney development Slit and Robo signaling restrict inappropriate migration of cells (Piper et al., 2000; Grieshammer et al., 2004; Yu et al., 2004). In fact, mutant mice lacking Slit2 do not develop a single ureteric bud, but rather, supernumerary ureteric buds that remain abnormally connected to the nephric duct (Grieshammer et al., 2004).
In adult rodent and human kidneys, Slit2 is expressed by many cell types, including vascular endothelial cells, glomerular endothelial, mesangial and epithelial cells, and tubular epithelial cells (Wu et al., 2001; Kanellis et al., 2004). In an animal model of crescentic glomerulonephritis, endogenous glomerular expression of Slit2 sharply decreased, promoting enhanced inflammation (Kanellis et al., 2004). When Slit2 was administered systemically in this short-term inflammatory model, renal function and renal histology improved significantly (Kanellis et al., 2004).
In U.S. Pat. No. 8,399,404, it was shown that, in a mouse model of IRI in which both renal pedicles are clamped, Slit2 and Slit2-N were shown to prevent renal failure due to acute kidney injury.
Chronic fibrosis after acute or repeated injury is not unique to the kidney, and results in conditions such as chronic obstructive lung disease, cardiomyopathy and heart failure, and liver cirrhosis. In all of these disorders, fibroblast activation occurs and the normal tissue becomes irreversibly replaced by fibrotic scar tissue.